Expression of prothymosin alpha in meiotic and post‐meiotic germ cells during the first wave of rat spermatogenesis

Abstract

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non-mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post-meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT-PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells.

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“…1) for either PTMA (using three different antibodies), the acrosomal region (using PNA lectin), or the sub‐acrosomal PT (using an antiserum raised against the protein extract from this region). PTMA fluorescence is consistent with the results previously obtained by imunocytochemical analysis (Ferrara et al, 2010): the peptide localizes in the antero‐dorsal region of eSPZ head, where it follows the curved shape of the cytoplasm, flanking 2/3 of the nucleus and eventually reaching the tip of the gamete, as equally evidenced with the three different antibodies (Fig. 1B,C).…”

“…The assembly of IAM proteins during the acrosome biogenesis shares its origin with those destined to the PT sub‐acrosomal layer. Interestingly, PTMA expression in adult rat testis perfectly matches (Ferrara et al, 2010). This is not the only intriguing factor: one of the most abundant PTMA‐binding proteins is karyopherin β1(Freire et al, 2001), a nuclear transport protein that is also located on the extravesicular side of the IAM (Tran et al, 2012).…”

“…In particular, to carry out our analysis, we chose two different markers: the lectin Arachis hypogaea agglutinin (PNA), which stains the acrosome (Kallajoki et al, 1985; Yoshida et al, 2010) and an antiserum raised against the protein extract from the PT, which labels the subacrosomal part of the PT (Oko et al, 2011). Although the features of the PT had led us to hypothesize a cross‐talk between its proteins and PTMA (Ferrara et al, 2010), our confocal analyses showed that PTMA signal overlapped with lectin staining, thus demonstrating PTMA localization in the acrosome, that is also a suitable environment for such an acidic protein.…”

“…Although PTMA specific role in reproduction is still to be elucidated, its involvement in spermatogenesis and its presence in male gametes has been borne out in lower Vertebrates like Torpedo marmorata (Prisco et al, 2009), Danio rerio (unpublished data) and Rana esculenta (Ferrara et al, 2009) as well as in the mammal R. norvegicus (Ferrara et al, 2010). The complex structural composition of mammalian sperm head on one side and the potential ubiquitous distribution of PTMA on the other made a thorough investigation on its sub‐localization necessary.…”

“…In 2010 our group reported the expression of Prothymosin α (PTMA) in rat testis (Ferrara et al, 2010). PTMA is a highly acidic small polypeptide; its association with male gametogenesis had been already described in non‐mammalian Vertebrates (Aniello et al, 2002; Ferrara et al, 2009; Prisco et al, 2009).…”